This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During the past few years mass spectrometry has emerged as leading technology for proteomics applications thanks to the continuous development of new methodologies and better instrumentation. Among these, a branch known as Structural Proteomics has grown into a powerful technique, capable of offering a wide variety of approaches to low resolution characterization of protein structure by combining protein chemistry with modern mass spectrometry. This approach has proven capable of delivering information that can be complementary to those obtained by high resolution techniques. Chemical crosslinking [1] is one of the most common methodologies employed in the analysis of protein complexes. In this work we explore limits and possibilities of a top-down [2,3] approach to the study of protein interactions and quaternary structure. Method: Lyophilized human hemoglobin (Sigma) was dissolved (10 [unreadable]M) in 10 mM sodium acetate buffer (pH 7.5). Aliquots of Bis[sulfosuccinimidyl]suberate (BS3) (5 mM, 10 mM, 25 mM, 50 mM, 100 mM) were added to different reaction mixtures, and 200-[unreadable]l aliquots were quenched with NH4HCO3 to a final concentration of 20 mM after 5, 15, 30, 60, 120 min. Samples were analyzed as is with a Thermo-Fisher LTQ-Orbitrap "Discovery" mass spectrometer using an Advion Triversa NanoMate ESI source. Tandem mass spectra were generated by LTQ-CID and HCD in the C-trap or nozzle-skimmer dissociation (NSD) and were deconvoluted using Xtract software (Thermo Scientific). Fragment mass lists were analyzed using BUPID-Topdown (Boston University Protein Identifier-Topdown), a custom-programmed software algorithm written in-house. Results: The first step of our analysis was the optimization of the crosslinking reaction between human hemoglobin and BS3 and of the sample preparation in order to render the mixture amenable to direct mass spectrometric (MS) analysis. The reagent employed, BS3, is a homobifunctional crosslinker that contains an amine-reactive N-hydroxysulfosuccinimide (NHS) ester at each end of an 11.4-A spacer arm. In order to assess the reaction kinetics and follow the formation different species, different reaction mixtures, with increasing molar excess of crosslinking agent, were followed by a time course analysis. With increasing reaction time, the samples showed a growing degree of complexity, and thus indicated the occurrence of a variety of combinations of protein modification and both intra- and intermolecular crosslinking events, ultimately leading to the disappearance of the sample signal. Among the analyzed mixtures, we were able to observe the formation of only homo-dimers of the alpha- and beta-subunits of hemoglobin, no hetero-dimer was ever detected. We isolated different charge states of the crosslinked homodimers and with the combination of different fragmentation techniques, e.g., LTQ-CID, HCD in the C-trap and nozzle-skimmer dissociation (NSD), applied to different charge states, we were able to locate the identify the specific amino acids involved in the crosslinking reaction, and thus the region of the protein involved in the interaction. From our MS/MS experiments we were able to observe that the covalent homodimer of the beta-subunits was formed via a crosslink between the K82 residues in both chains, suggesting that, in the native tetrameric structure of hemoglobin, these two residues are located close enough in space to allow the formation of a covalent crosslink. Similar results were found for the alpha-subunits. In this case we were able to locate the covalent crosslink between the N-terminal amino group of one subunit with either the K127 or K139 of another. No fragment has yet been found that can distinguish between these two lysines. Discussion: Our results are consistent with X-ray structural determinations of hemoglobin. They also show that the well established technique of protein crosslinking for conformational analysis can be performed by a top-down methodology. With this approach we were not only able to locate the regions of protein interaction by direct analysis of protein, but also to analyze the reaction behavior in order to choose the optimal reaction conditions of lowest amount of reagent and time of reaction, in order to preserve the native tertiary structure of the protein under analysis. This methodology no only offers the possibility for quaternary structural analysis of proteins even when crystals are not available, but also expedites the normal procedures employed for chemical crosslinking, including gel based analysis that requires larger amounts of sample and offer a lesser degree of control over the reaction conditions. 1.Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions. A. Sinz, Mass Spectrom Rev. 2006 25(4):663-82 2.Top down'versus 'bottom up'protein characterization by tandem high-resolution mass spectrometry. Kelleher NL, Lin HY, Valsakovic GA, Aaserud DJ, Fridrikson EK, Beavil A, Holowka D, Gould HJ, Baird B, McLafferty FW. J Am Chem Soc 1999, 121:806[unreadable]812 3.A top down approach to protein structural studies using chemical cross-linking and Fourier transform mass spectrometry. Kruppa GH, Schoeninger JS, Young MM. Rapid Commun Mass Spectrom 2003, 17:155[unreadable]162